Abstract: Microbial glycans are critical for viability and virulence, and they mediate interactions of microbes with other organisms, including their hosts. The benefits of understanding microbial glycan structure and function are wide-ranging, including the development of new antimicrobial therapies, novel strategies to control chronic autoimmune conditions (e.g., Crohn?s disease, ulcerative colitis, asthma), and innovative approaches to enhance health through modulating microbiome composition. A major impediment to achieving these benefits is the difficulty of identifying the glycans present on an organism and elucidating microbial glycan function. Microbial glycans contain carbohydrate building blocks absent from their mammalian counterparts, and glycan function depends on these orthogonal molecular features. However, the majority of tools available for glycan analysis are designed for mammalian glycans. As a result, the analysis of microbial glycans depends on specialized expertise in glycobiology and is therefore inaccessible to the broad scientific community. The goal of this application is to address the need for facile tools by generating a curated set of probes for detecting, isolating, and interrogating the function of microbial glycans and the microbial species upon which they are presented. We plan to generate these microbial glycan analysis probes (mGAPs) from 20 human humoral lectins, which mediate immune defense through microbial glycan engagement. Aim 1 focuses on generating mGAPs using versatile ligation strategies, including biotin ligase and sortase-mediated conjugations. Together these approaches provide the means to site-specifically append diverse reporter groups or other functionality for user-defined applications. The experiments in Aim 2 involve benchmarking the microbial glycan binding specificity of the mGAPs to render these tools maximally useful to researchers in the field. Aim 3 focuses on testing mGAP-based lectins as tools to investigate microbial community composition, as mGAPs have the requisite attributes for multiplexing and high-throughput analysis. Finally, mGAP kits will be assembled and disseminated to researchers to probe lectin and microbial glycan structure and function.